protein extraction from human tear using schirmer strip

גילוי סמנים ביולוגיים לא פולשניים לפרוטאומיקה של דמעות באמצעות LC-MS

Tear proteomics has received attention to explore potential biomarkers for ocular diseases and conditions1,2,3,4,5,6, to access the pathogenesis of the ocular and systemic condition, as well as to exploit the advantage of non-invasive tear sample collection using Schirmer strips. The technological advancement of next-generation mass spectrometry has enabled protein quantification in microliter-scale tears with accuracy and precision not possible in the past. Sample preparation methods are not yet standardized. A robust and standardized sample preparation workflow is essential for the success of clinical application in tear protein biomarker research. The suspension trapping (S-Trap) sample preparation workflow was recently reported as an effective and sensitive sample preparation method for broad downstream proteomic analysis7,8. Yet, this strategy has not been well-reported in the analysis of human tear proteome, outperformed filter-aided sample preparation (FASP) and in-solution digestion in terms of enzymatic digestion efficiency and the greater number of protein identification by mass spectrometric analysis9. The S-Trap-based approach was demonstrated in the preparation of retinal tissue 10, formalin-fixed, paraffin-embedded (FFPE) tissue11, cells12, microorganism13, and liquid biopsies14,15.
This protocol describes an integrated quantitative workflow from clinical samples to enzymatically digested protein for the discovery of a non-invasive tear protein biomarker panel with a rapid, reproducible, and robust technical strategy. Briefly, tear fluid was collected using a standard Schirmer strip and immediately dried with an ophthalmic frame heater to prevent protein autolysis at room temperature. Embedded total proteins were extracted using 5% sodium dodecyl sulfate (SDS) lysis buffer according to the manufacturer&#39;s suggestion, followed by protein assay measurement. The extracted lysate then underwent standard reduction with dithiothreitol (DTT) and alkylation with iodoacetamide (IAA).
After acidification with phosphoric acid, suspension trapping column protein precipitation buffer containing 90% methanol and 100 mM triethylammonium bicarbonate (TEAB) was added to aggregate proteins. The sample was then transferred into a new suspension trapping micro column. Enzymatic digestion with done with sequencing grade trypsin at a 1:25 ratio (w/w, trypsin: protein) at 47 &#176;C for 1 h. The resulting peptides were then eluted via centrifugation, sequentially with 50 mM TEAB, 0.2% aqueous formic acid (FA), and 50% acetonitrile (ACN) containing 0.2% FA. The eluted peptides were vacuum-dried and reconstituted in 0.1 % FA. The peptide concentration was measured and adjusted to 0.5 &#181;g/&#181;L for mass spectrometric analysis.

מחבר זרקור: קידום ניתוח נוזל הדמעות באמצעות פרוטוקול סטנדרטי לחקר פרוטאומיקה 

הכנת דגימה ללכידת תרחיף חלבונים עבור פרוטאומיקה של דמעות על ידי כרומטוגרפיה נוזלית-ספקטרומטריית מסה טנדם

Our research aims to demonstrate the use of a protein suspension trapping sample preparation, known as S trap for mass spectrometry-based research. This protocol employs a rapid, reproducible, and standardized sample preparation of human tear fluid sample collected using the for translational research in ophthalmology. Human tear fluid is a body fluid that can be collected for non-invasive molecular tests in ophthalmology.There is a lot of standardized methods of tear fluid collection and sample preparation, which are critical to successful clinical research of diseases and conditions. Non-standardized methods are difficult to compare and interpret between methodologies and therefore affect chemical outcomes. This protocols allows tear body fluid sampling and storage at room temperature.This helps with easier sample collections and shipping. It provides a rapid and robust peptide extractions for mass spectrometer analysis, which improves peptide recovery and identifications. This protocol has minimal sample preparation steps to minimize error and uncertainties.The fast and superior performance of protein identification and quantification by mass spectrometry will facilitate clinicians and researchers in the development of non-invasive molecular tests. Optimize the sample preparation will always be one of the critical steps. Regardless of the detection method, the clinical value of human tear fluid is yet to be fully investigated.The proposal sample preparation method would hopefully open the door for potential clinical application in the near future.

Watch this Scientific Journal Video about Protein Extraction from Human Tear Using Schirmer Strip at JoVE.com

Protein Extraction from Human Tear Using Schirmer Strip  

מיצוי חלבון מדמעות אדם באמצעות רצועת Schirmer

התחילו בחיתוך והשלכת הקצה הקדמי של רצועת שירמר המכיל את הדמעות האנושיות המיובשות מעבר לאפס מילימטר. לאחר מכן חותכים את הרצועה למרווחים של מילימטר אחד ומניחים את החתיכות בצינור מיקרו צנטריפוגה של 1.5 מיליליטר. לאחר מכן, הוסף 100 מיקרוליטר של חיץ ליזיס לצינור מיקרו צנטריפוגה.מערבבים אותו ב-1000 סל"ד בטמפרטורת החדר למשך שעה במיקסר תרמי. לאחר צנטריפוגה קצרה, מעבירים את הסופרנאטנט לצינור מיקרו-צנטריפוגה חדש בקוטר 1.5 מיליליטר. בעזרת מלקחיים נקיים, מעבירים את הרצועות לקצוות פיפטה של 200 מיקרוליטר.החזירו את הקצוות לאותו צינור מיקרו-צנטריפוגה, ולאחר מכן בצעו צנטריפוגות לשחזור דגימה מקסימלי. מדוד את ריכוז החלבון באמצעות חומצה ביכונית, או בדיקת חלבון תואמת.

protein-extraction-from-human-tear-using-schirmer-strip

Research

JoVE Journal

Medicine

Protein Extraction from Human Tear Using Schirmer Strip

429 Views.  The Hong Kong Polytechnic University. התחילו בחיתוך והשלכת הקצה הקדמי של רצועת שירמר המכיל את הדמעות האנושיות המיובשות מעבר לאפס מילימטר. לאחר מכן חותכים את הרצועה למרווחים של מילימטר אחד ומניחים את החתיכות בצינור מיקרו צנטריפוגה של 1.5 מיליליטר. לאחר מכן, הוסף 100 מיקרוליטר של חיץ ליזיס לצינור מיקרו צנטריפוגה.

Video: Protein Extraction from Human Tear Using Schirmer Strip  

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.

This protocol describes a method for collecting tear samples using Schirmer strips and an integrated quantitative workflow for discovering non-invasive tear protein biomarkers. The suspension trapping sample preparation workflow enables fast and robust tear sample preparation and mass spectrometric analysis, resulting in higher peptide recovery yields and protein identification than standard in-solution procedures.

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for ...

Collection of Human Tear Fluid Using Schirmer Strip

Begin by wearing gloves and sanitizing hands to prevent sample contamination. Check that the inner packaging of the Schirmer strip is sterile, unexpired, and intact. Next, bend the top of the Schirmer strip slightly inward at the zero millimeter mark near the semicircle tip.Remove the strip by holding the end avoiding direct contact with the zero to 25 millimeter sample collection area. Instruct the subject to look away. Then gently pull down the lower eyelid.Hook the strip end in the lower fornix near the lateral canthus region. After repeating the process for the other eye, ask the subject to close their eyelids gently to minimize irritation. Collect the tear fluid for approximately five minutes until a 15 to 20 millimeter tear sample is obtained.Instruct the subject to open their eyes. Gently pull down the lower eyelid and remove the strips. Next, inspect the hydrated length on the strip and record the collected amount in millimeters.Dry the strip using a standard clean frame heater until the fluid evaporates. Once dried, place the Schirmer strip into a labeled cryo tube and store it in a cool dark place for further processing.

איסוף נוזל דמעות אנושי באמצעות רצועת שירמר

Begin by cutting and discarding the front end of the Schirmer strip containing the dried human tears beyond the zero millimeter mark. Then cut the strip into one millimeter intervals and place the pieces in a 1.5 milliliter micro centrifuge tube. Next, add 100 microliters of lysis buffer to the micro centrifuge tube.Vortex it at 1000 RPM at room temperature for one hour in a thermo mixer. After brief centrifugation, transfer the supernatant to a new 1.5 milliliter micro centrifuge tube. Using clean forceps, move the strips into 200 microliter pipette tips.Place the tips back into the same micro centrifuge tube, then centrifuge it for maximum sample recovery. Measure the protein concentration using bicinchoninic acid or a compatible protein assay.

Suspension-Trapping Sample Preparation of Human Tear Protein

Begin by normalizing the human tear protein samples to 50 micrograms of protein. Then add 200 millimolar dithiothreitol, or DTT, to a final concentration of 20 millimolar DTT, incubated at 95 degrees Celsius for 10 minutes. After cooling the protein solution to room temperature, add 400 millimolar iodoacetamide to a final concentration of 40 millimolar, then incubate in the dark at room temperature for 10 minutes.Next, add 12%aqueous phosphoric acid in a one to 10 ratio, resulting in a 1.2%final concentration. Vortex the mixture briefly to mix the solution. Add protein binding buffer in a one to six ratio before vortexing again.Uncap the suspension trapping micro spin column and assemble the spin column onto a two milliliter microcentrifuge tube to collect flowthrough. Add up to 200 microliters of acidified protein lysate mixture into the column. Centrifuge the column at 4000 G for 20 seconds.Next, add 150 microliters of protein binding buffer and centrifuge as before to wash the suspended protein. Assemble the suspension trapping micro column onto a new 1.5 milliliter microcentrifuge tube. Carefully add 20 microliters TEAB digestion buffer onto the filter inside the spin column, avoiding bubbles and air gaps.Securely cap the suspension trapping micro spin column to prevent evaporation. Incubate at 47 degrees Celsius for one hour in a thermal mixer without shaking. Elute peptides with 40 microliters of 50 millimolar TEAB.Then centrifuge it at 4000 G for 20 seconds. Now add 40 microliters of 0.2%formic acid and centrifuge as before. Lastly, add 35 microliters of 0.2%formic acid and 50%to acetonitrile before centrifuging again.Pull the three eluents and dry them using a vacuum centrifuge. Once dried, store the samples at 80 degrees Celsius for future analysis. The data dependent acquisition search yielded a total of 1183 plus or minus 118 proteins in the suspension trapping group, and 874 plus or minus 70 proteins at 1%FDR in the in-solution group.Gene ontology analysis revealed similar proteomes for both approaches with main function as binding catalytic activity and molecular function regulation.